Thienyl-penicillins



Patented July 31, 1951 UNITED STATES PATENT OFFICE THIENY L-PENICILLIN SOtto K. Behrens, Reuben G. Jones, and Quentin F. Soper, Indianapolis,Ind, and Joseph W. Corse, Lafayette, Calif., assignors to Eli Lilly andCompany, Indianapolis, Ind., a corporation of Indiana N Drawing.Application May 3, 1949, Serial No. 91,216

8 Claims. (Cl. 260-239.1)

produced as metabolic products by a penicillinproducing mold. Thecomplete molecular structures of these compounds had not been definitelyestablished when said prior applications were filed but suflicientelucidation of structure had been accomplished to allow the assignmentto them of the following empirical formula:

in which R0 has the same meaning as set forth above.

We have made the surprising discovery that a penicillin-producing moldmay be induced to produce a novel penicillin, by incorporating in thenutrient medium wherein the mold is grown, a .selected organic compound,called herein a precursor compound. Such selected precursor compound,although foreign to the normal metabolic requirements of the mold, maybe metabolized and incorporated in substantial part in the molecule of anovel penicillin. This result is especially surprising in view of therecognized specificity of the enzyme systems whereby lower organismsmaintain growth and development. It is further surprising that use of aselected precursor com- 'pound may lead to the production of. a novelpenicillin to the substantial exclusion of the known andnormally-produced penicillins.

The present invention contemplates novel prod-,- ucts of fermentativeprocesses which comprise growing a penicillin-producing mold inassociation with a culture medium containing nutrient material and aselected precursor compound, said product as produced consistingessentially of a penicillin represented by the formula in which Rrepresents; a thienyl radical. The thienyl radical may be either2-thieny1 or 3-thi.- enyl, and may be substituted or unsubstituted. Thesubstituted thienyl radicals which R represents include S-methylthienyland the 5-halothienyl radicals.

The novel penicillins are ordinarily produced in the form of a salt, forexample the sodium salt of the penicillin acid. The salt may be eitherthe salt originally produced or a difierent salt to which the originalproduct is transformed, and preferably is one of the salts ordinarilyemployed in the administration of the known penicillins, such as thecommon metal salts, for example, the sodium salt, the potassium salt,the ammonium salts, the calcium salts, etc;

According to the present knowledge of the structure of penicillin, theproducts of this application, as defined above, including both acids andsalt thereof, may be represented by the following structural formula:

in which R has the same meaning as before, and in which X representshydrogen when the penicillin is in acid form and represents asalt-forming radical when the penicillin is in salt form, suchsalt-forming radicals including those customarily employed in'penicillinproduction and therapy, such as potassium, sodium, calcium, ammonium,etc.

For convenience, we use below the empirical formula, e. g., -CmH13N2O4S,as representing the molecular portion of the penicillin to which thethienyl radical is attached.

The new penicillins may be named by reference to the particular thienylradical contained therein. Thus, for example, a penicillin wherein thethienyl radical is the 2-thienyl radical, is named Z-thienyl-penicillin(or a-thienyl-penicillin), and a penicillin wherein the thienyl radicalis the 5- 3 methyl-3-thienyl radical, is called 5-methyl-3-thienyl-penicillin.

Broadly speaking, a method of producing a novel penicillin in accordancewith this invention is as follows: There is provided a nutrient mediumsuitable for the growth of a penicillinproducing mold. To the nutrientmedium is added in effective amount a precursor compound comprising anR-monosubstituted acetic acid. Precursor compounds useful for thispurpose comprise monosubstituted acetic acids represented by the formulawherein R has the same meaning as before. In place of themonosubstituted acetic acids, there may be used equivalents of suchacetic acids, said equivalents comprising those compounds readilyconverted by the mold to the monosubstituted acetic acids. Suchequivalents include simple derivatives of the acids such as their salts,esters, amides, and anhydrides, and may include other compounds, whichthe mold may convert to the monosubstituted acetic acids, such asw-R-Sllbstituted, saturated straight-chain alcohols, amines, aldehydes,and acids containing an even number of carbon atoms, and the simplederivatives thereof.

The culture medium composition comprising nutrient material andprecursor compound is inoculated with a penicillin-producing mold andthe mold is grown under penicillin-producing conditions, during whichgrowth a new penicillin is produced by the mold as a metabolic product.After mold growth, the mold mycelium is separated from the culturemedium, and from the latter the novel penicillin is separated.

The isolation of the new penicillin may be effected by methods known tothe art, such as adsorption and extraction, to obtain a productsufficiently pure for practical purposes. If a purer product is desired,the new penicillin may be subjected to additional methods ofpurification such as partition chromatography and elution, andrecrystallization.

The novel penicillin desirably is recovered in the form of one Of itssalts, for example the sodium or potassium salt. Identification of thenovel penicillin may be confined by methods known to the art, suchmethods comprising analysis, spectroscopic absorption, X-ray diffractionand antibacterial tests.

The nutrient material used in the composition wherein the mold is grownmay comprise ingredients such as water, sugars, inorganic salts anddesirably one or more indeterminate compositions such as corn steepamino acids and bran. Numerous suitable nutrient media comprisingmaterials of the type mentioned are known to the art.

During the growth of the mold the culture medium comprising nutrientmaterial and precursor compound is maintained at a suitable temperature,for example, in the range of 20-30 C. The range of temperature which hasbeen found to be particularly suitable is from 24-26 C. The period oftime during which the mold is grown will depend upon the objectivedesired. Thus the mold may be grown only during the period of itsmaximum rate of growth before mold growth is interrupted preliminary toisolating the new penicillin. Such a period generally is from 2 to 3days. On the other hand, the mold may be grown for a longer period oftime to obtain the maximum yield of new penicillin.

In such a case, mold growth is usually continued for about 4 to 5 days.

The mold may be grown under various conditions. For example, the moldmay be grown without agitation of the culture medium, in which case themold grows on the surface of the medium. Alternatively, the culturemedium may be agitated by shaking or stirring during the growth of themold in which case the mold is dispersed throughout the culture mediumand grows while so dispersed.

The molds suitable for the purposes of this invention are mold organismsof the type capable of producing penicillins. Such organisms includemolds of the Penicillium notatum-chrysogenum group as well as certainmolds of the Aspergillus group. It is to be understood that not all moldstrains are equally efiicient for the purposes of this invention. By wayof example, mold strains suitable for the purposes of this invention arethose known as strains X1612 and Q176 of the Penicilliumnotatum-chrysogenum group and strain G147 of the Aspergillus ,flavusgroup.

The concentration of the precursor compounds employed in the culturemedium may vary over a substantial range. The precursor compounds may bepresent in the culture medium in concentrations of the order of about 1percent, but it is usually desirable that smaller concentrations beemployed since there is no particular advantage to be gained inemploying concentrations in substantial excess of those necessary toproduce the optimum efiect. It appears at present that the optimumconcentration of the monosubstituted acetic acids and derivativesthereof lies in the range of about 0.01 to about 0.05 percent on aweight volume basis when mold strain X1612 is used, and that thisoptimum concentration may range upwardly when mold strain Q176 is used.

The precursor compound may be associated with the mold and culturemedium at any suitable time. Thus the materials of the nutrient mediummay be inoculated with the mold and the precursor compound to beemployed may be incorporated either before or after inoculation of theculture medium with the mold.

The following specific examples further illustrate the invention.

Example 1 The sodium salt of a-thienyl-penicillin represented by theformula HO OH a I -CmHnNaOaSNa s can be prepared in the followingmanner:

A culture medium is prepared in the following proportions portions in 1liter Erlenmeyer flasks, sterilized, inoculated with a spore suspensionof Penicillium mold, strain N. R. R. L. 1976, and stoppered with cottonplugs. The flasks are maintained at a temperature of about 23-26 C. andshaken consteamy for fivedays'. The"fiask'-contents are then filtered toremove the mold mycelium, the filtrate cooled. to about 0 C., acidifiedto about pH 2.2 with orthophosphoric acid and shaken with about one halfits volume of amyl acetate. The amyl acetate layer is separated andextracted with three 100 cc.;portions ,of ,cqld water to which cold N/.sodii mbicarbonate solu i is added during the courseof each extractionuntil a pH of about -7.1--to 7.3 is attained in the aqueousphase. Theaqueous extracts are combined, cooled to'about 0 C., acidified to aboutpH 2.2 with orthophosphoric acid and extracted with three 100 cc.portions of ether. The ether freezing and evaporation in vacuo from thefrozen state. I T

The resulting dry sodium salt of the a-thienylpenicillin is washedseveral times with anhydrous acetone. The sodium salt is thencrystallized by dissolving it in 2 cc. of 90 percent. acetone andreprecipitating it with 2 cc. of anhydrous acetone. 213 mg. of thesodium salt of a-thienyl-penicillin is obtained.

The sodium salt of c-thienyl-penicillinprepared according to the aboveprocedure assayed about 1685 Oxford units per mg. when tested againstStaph. aureus strain 209P. A differential assay carried out on Staph.aareus, strain extracts are scombined, .dried over magnesium l5 209P,and B. sabtilis, strain N. R. R. L. B.-558, sulfate and arethen passedthroughaehromatogave a value of about 1.13. The optical rotation graphictype silica adsorption"coliiinii about 30 was foundtobe mm. in diameterand 300 mm. long, and contain- 0 ing a pH 6.2"phos1zw'hate bufieri L-Thesilica [a] 265 column-is developed by percolation with-.six 100 D QC! Qm S d .9t1lQ Y QMaiIl s as a 0.3 percent solution in water. Analysiscreasmlg amounts of methanol m the order of showed the presence of 46.46percent carbon, 3.81 1 21/2 percent f percent hydrogen, 7.40 percentnitrogen and 17.18

The developed s 1hca column q q Into percent sulfur as compared with thecalculated about 12 equal sections and each section is eluted values of46.40 percent carbon, 417 percent Wlth three portlons of M/15 phosphatedrogen, 7.73 percent nitrogen and 17.69 percent buffer of pH 7.0. Theeluates are assayed bacsulfim teriologically to determine theirpenicillin con- Example 2 tent. 91 percent of th total antibioticactivity possessed by all the eluates originates in a single 30 Otherthienyl-penicillins of our invention are band in the silica column andresults from the prepared by the procedure of the foregoing expresenceof a-thienyl-penicillin. This band 00- ample, using precursorscontaining the thienyl cupies a position similar to that in whichPeniradical desired in the new pencillin. Thus, for cillin G is found incomparable columns. The example, the following new thienyl-penicillinseluates obtained from the silica gel sections are prepared using theprecursors listed:

New Penicillin Precursor For The New Penicillin which make up thisuniform band are combined, cooled to about 0 C., acidified to about pH2.2 with orthophosphoric acid and extracted with three cc. portions ofchloroform. The combined chloroform extracts are then passed through asilica adsorption column containing a pH 6.2 phosphate bufier. Thissilica gel column is developed by percolation with three 100 cc.portions of chloroform containing successively increasing amounts ofmethanol in the order of 1, 2 and 3 percent. The developed silica columnis then divided into 12 equal sections and each section is eluted withthree 30 cc. portions of M/15 phosphate buffer of pH 7.0.Bacteriological assay of the eluates shows 95 percent of the totalantibiotic activity concentrated in a single band in the silica column.The eluates obtained by extraction of the silica column sections whichcomprise this band are combined, cooled to about 0 C., acidified toabout pH 2.2 and extracted with three 100 cc. portions of ether. Theether extracts are combined and extracted with about 75 cc. of a colddilute aqueous solution of sodium hydroxide to which N/ 10 sodiumhydroxide solution is added during the course of the extraction so thata final pH of about 7.0 is obtained in the aqueous phase. From thisaqueous solution the sodium salt of a-thienyl-penicillin may be isolatedby any suitable means, for example, by

Example 3 The new penicillins obtained in the form of their sodium saltsmay be converted to other salts in a number of ways. One way is asfollows: An aqueous solution of the sodium salt, acidified to about pH2, is extracted with an equal volume of an organic solvent, such as amylacetate, ether, chloroform, or the like. The organic solvent solution,containing the new penicillin in acid form, is extracted with an aqueoussolution containing the cation of the desired salt, for example, asolution of potassium hydroxide, calcium phosphate, ammonium hydroxide,or the like, at about pH 8.5. The aqueous extract contains thepenicillin as the corresponding salt, for example, the potassium salt,the calcium salt, or the ammonium salt of the penicillin, and such saltis suitably recovered from the solution, as by drying in vacuo from thefrozen state.

We claim as our invention:

1. A compound of the group consisting of a new penicillin acid and itssodium, potassium, calcium and ammonium salts, said acid beingrepresented by the formula OOH in which R is a member ofthe groupconsisting. 5. A new penicillin represented by the formula of hydrogen,methyl and halogen. 0 s

2. A new penicillin represented by the formula U A \Gwm) U i 5 s halogens cHze-qm-cn-o C GHQZ 8 420015! N H 6. Z-thienyl-penicillin.

1' 5 'z. 5-bromo-2-thienyl-penicillin. 8. 5- -2- h 1- eni 11in.

3. A new penicillin represented by the formula chlom t ieny 8. BEHRENSREUBEN G. JONES.

s CH1PJ-NHCHC owHm QUENTIN F. SOPER.

l H JOSEPH W. CORSE. s F) (10011 p REFERENCES CITED 4. A new penicillinrepresented by the formula The following references are of record in the0 s file of this patent: I cH,i' NH oE e ownm I UNITED STATES PATENTS H20 Number Name Date ll I l OH 2,479,297 Behrens et all Aug. 16. 1949

1. A COMPOUND OF THE GROUP CONSISTING OF A NEW PENICILLIN ACID AND ITSSODIUM, POTASSIUM, CALCIUM AND AMMONIUM SALTS, SAID ACID BEINGREPRESENTED BY THE FORMULA